ABSTRACT Influenza A virus (IAV) infected patients admitted to the ICU face severe secondary infection complications. There is increasingly strong evidence that secondary fungal infections with Aspergillus fumigatus (Af) present a significant risk for ICU patients with severe IAV infection. Overall, approximately 20% of severe IAV patients in the ICU may develop secondary Af infection. Currently, there is a critical gap in understanding how and why these fungal secondary infections develop in IAV patients. This R21 application aims to fill this gap by generating a robust small animal IAV-Af coinfection model that can be used to dissect the molecular mechanism(s) by which IAV infection increases susceptibility to Af challenge. Our initial pilot experiments demonstrate that IAV infection four days prior to Af challenge results in significant fungal growth and tissue invasion, which is absent in nave mice challenged with Af. In SA1 of this R21 proposal we will further optimize and develop this IAV-Af coinfection model to ensure it is highly reproducible and define the importance of IAV serotype and Af clinical strain heterogeneity in susceptibility to coinfection. In SA2, we will test the hypothesis that IAV infection impairs pulmonary leukocyte anti-fungal activity using two innovative techniques: 1) Fluorescent Aspergillus reporter (FLARE) strains to directly quantify anti-fungal activity of leukocyte populations at a cell single level and 2) Single cell RNA sequencing (scRNA-Seq) to monitor transcriptional changes in pulmonary leukocyte populations in an unbiased manner. These data will provide the first insights into mechanisms driving Af susceptibility following IAV infection. Overall, this research fills a critical gap by providing the field with a unique and highly needed IAV-Af coinfection model and provides a unique and robust dataset for the development of future focused, mechanistic R01 applications.